Project Summary The clinical efficacy of immune checkpoint blockade (ICB) in a subset of metastatic melanoma patients has catalyzed a revolution in cancer care leading to its application to a variety of other tumor types. The discrepancy in response to ICB between, and even within, tumor types, however, indicates that additional mechanisms of suppression must be targeted to expand utility of these therapies. Recent biomarker studies, indicate that presence of cytotoxic T cells (CTLs) within tumor microenvironments enriches for patients who are likely to respond, suggesting that efforts to improve intratumoral accumulation of tumor-specific CTLs might enhance response to therapy. The current proposal will examine mechanisms regulating T cell exit from melanoma and propose inhibition of egress as a novel strategy to improve T cell retention and thus response to ICB. We will test the hypothesis that tumor-associated lymphatic vessels influence intratumoral CTL pools by directing their egress. Transgenic mice expressing a photoconvertible protein will be used to in situ label and in vivo track tumor resident lymphocyte populations. Using this model system paired with transgenic T cells and altered antigen ligands, we will determine the role antigen encounter plays in determining which cells egress from melanoma microenvironments to delineate how T cell egress from tumors contributes to the diversity of the intratumoral T cell repertoire. Our preliminary data indicates that T cells specific for tumor antigens egress from tumors indicating that inhibition of tumor exit, to retain these T cells, might improve response to immunotherapy. To test this, we will determine the functional significance of candidate chemokine receptors, CXCR4 and CXCR6, which we predict regulate T cell egress and retention respectively. We will determine the therapeutic synergy of combining agents targeting these pathways with immune checkpoint blockade. Finally, we have shown that disrupting lymphatic vessel crosstalk with CTLs, mediated by IFN?, improves tumor control. We will explore the role of IFN?-mediated bidirectional crosstalk in regulating T cell egress through lymphatic vessels and thus propose T cell egress as a mechanism of immune resolution coopted by tumor microenvironments to mediate immune evasion. Taken all together, we propose that CTLs integrate multiple signals in melanoma microenvironments that tune their accumulation and function in concert with the lymphatic vasculature. We predict that inhibition of CTL egress represents a tractable clinical strategy that can synergize with therapies targeting additional immunosuppressive mechanisms (e.g. metabolism and exhaustion), and mechanistic studies described herein will provide the rationale for future clinical translation.